Bottom Line:
Phylogenetic analysis of the chickpea and well-known stress-related Arabidopsis and rice NACs enabled us to predict several putative stress-related CaNACs.Nine-teen of the 23 CaNACs examined were found to be dehydration-responsive in chickpea roots and/or leaves in either ABA-dependent or -independent pathway.Our results have provided a solid foundation for selection of promising tissue-specific and/or dehydration-responsive CaNAC candidates for detailed in planta functional analyses, leading to development of transgenic chickpea varieties with improved productivity under drought.

ABSTRACTThe plant-specific NAC transcription factors (TFs) play important roles in regulation of diverse biological processes, including development, growth, cell division and responses to environmental stimuli. In this study, we identified the members of the NAC TF family of chickpea (Cicer arietinum) and assess their expression profiles during plant development and under dehydration and abscisic acid (ABA) treatments in a systematic manner. Seventy-one CaNAC genes were detected from the chickpea genome, including 8 membrane-bound members of which many might be involved in dehydration responses as judged from published literature. Phylogenetic analysis of the chickpea and well-known stress-related Arabidopsis and rice NACs enabled us to predict several putative stress-related CaNACs. By exploring available transcriptome data, we provided a comprehensive expression atlas of CaNACs in various tissues at different developmental stages. With the highest interest in dehydration responses, we examined the expression of the predicted stress-related and membrane-bound CaNACs in roots and leaves of chickpea seedlings, subjected to well-watered (control), dehydration and ABA treatments, using real-time quantitative PCR (RT-qPCR). Nine-teen of the 23 CaNACs examined were found to be dehydration-responsive in chickpea roots and/or leaves in either ABA-dependent or -independent pathway. Our results have provided a solid foundation for selection of promising tissue-specific and/or dehydration-responsive CaNAC candidates for detailed in planta functional analyses, leading to development of transgenic chickpea varieties with improved productivity under drought.

pone-0114107-g006: Expression of selected CaNAC genes in chickpea roots under dehydration and ABA treatments.Expression data were obtained by RT-qPCR of treated (ABA or dehydration) and well-watered (WW) control root samples collected at indicated time points. Expression data were obtained by RT-qPCR of collected root samples. Mean relative expression levels were normalized to a value of 1 in water-treated control root samples. Error bars = SE values of three biological replicates. Asterisks indicate significant differences as determined by a Student's t-test (*P<0.05; **P<0.01; ***P<0.001). Membrane-bold CaNACs are underlined.

Mentions:
Using the criterion of fold-change ≥2 and P<0.05, the majority of the tested CaNACs were found to be dehydration-responsive in leaves and/or roots of chickpea plants (Figure S3A). Among the 23 CaNACs, 14 genes, of which 3 CaNTLs (CaNTL2/CaNAC19, CaNTL5/CaNAC41 and CaNTL7/CaNAC57), were up-regulated, whereas only 4 genes, of which one CaNTL (CaNTL1/CaNAC04), were down-regulated by at least 2-fold in leaves after 2 and/or 5 h of dehydration (Figure 5, Figure S3A). CaNAC06 and CaNAC67 were the two most highly induced genes (over 200- and 300-fold, respectively), whereas CaNAC02 and CaNAC04 were the two most significantly repressed genes (23.8-fold and 28.6-fold, respectively after 5 h of dehydration) in chickpea leaves by dehydration. As for the roots, 12 genes, of which 2 CaNTLs (CaNTL2/CaNAC19 and CaNTL6/CaNAC44), were induced, whereas 3 genes, of which one CaNTL (CaNTL1/CaNAC04) were repressed by at least 2-fold after dehydration for 2 and/or 5 h (Figure 6, Figure S3A). In comparison with dehydrated leaves, the degree of induction in dehydrated roots was approximately 10-fold lower, with the highest induction of ∼23-fold recorded for CaNAC67 at 5 h of dehydration. Thus, CaNAC67 being induced the most highly in both tissues is a promising candidate gene which deserves further and in-depth in planta molecular and functional analyses under drought. A number of studies have indicated that TF encoding genes with high inducibility by stress are preferable for selections of further in planta functional studies as they might have potential for development of improved stress-tolerant transgenic plants by overexpression approach [58], [66]. Additionally, the repression degree of CaNACs in dehydrated roots versus dehydrated leaves was also lower by approximately 4-fold. For instance, CaNAC02 was the most highly down-regulated by dehydration in roots with a fold-change of only 6.2. In addition, CaNAC24 was deserved to be mentioned as this gene was induced (2.6-fold and 3.7-fold at 2 and 5 h after dehydration, respectively) in dehydrated roots but repressed (3-fold at 2 h of dehydration) in dehydrated leaves. It would be then interesting to study how CaNAC24 is involved in regulation of chickpea responses to drought. We hypothesize that under drought stress the up-regulation of CaNAC24 in roots might contribute to enhancement of root development, whereas its down-regulation in leaves might contribute to repression of leaf and/or shoot growth. These changes would enhance the adaptation of chickpea plants under limited water conditions.

pone-0114107-g006: Expression of selected CaNAC genes in chickpea roots under dehydration and ABA treatments.Expression data were obtained by RT-qPCR of treated (ABA or dehydration) and well-watered (WW) control root samples collected at indicated time points. Expression data were obtained by RT-qPCR of collected root samples. Mean relative expression levels were normalized to a value of 1 in water-treated control root samples. Error bars = SE values of three biological replicates. Asterisks indicate significant differences as determined by a Student's t-test (*P<0.05; **P<0.01; ***P<0.001). Membrane-bold CaNACs are underlined.

Mentions:
Using the criterion of fold-change ≥2 and P<0.05, the majority of the tested CaNACs were found to be dehydration-responsive in leaves and/or roots of chickpea plants (Figure S3A). Among the 23 CaNACs, 14 genes, of which 3 CaNTLs (CaNTL2/CaNAC19, CaNTL5/CaNAC41 and CaNTL7/CaNAC57), were up-regulated, whereas only 4 genes, of which one CaNTL (CaNTL1/CaNAC04), were down-regulated by at least 2-fold in leaves after 2 and/or 5 h of dehydration (Figure 5, Figure S3A). CaNAC06 and CaNAC67 were the two most highly induced genes (over 200- and 300-fold, respectively), whereas CaNAC02 and CaNAC04 were the two most significantly repressed genes (23.8-fold and 28.6-fold, respectively after 5 h of dehydration) in chickpea leaves by dehydration. As for the roots, 12 genes, of which 2 CaNTLs (CaNTL2/CaNAC19 and CaNTL6/CaNAC44), were induced, whereas 3 genes, of which one CaNTL (CaNTL1/CaNAC04) were repressed by at least 2-fold after dehydration for 2 and/or 5 h (Figure 6, Figure S3A). In comparison with dehydrated leaves, the degree of induction in dehydrated roots was approximately 10-fold lower, with the highest induction of ∼23-fold recorded for CaNAC67 at 5 h of dehydration. Thus, CaNAC67 being induced the most highly in both tissues is a promising candidate gene which deserves further and in-depth in planta molecular and functional analyses under drought. A number of studies have indicated that TF encoding genes with high inducibility by stress are preferable for selections of further in planta functional studies as they might have potential for development of improved stress-tolerant transgenic plants by overexpression approach [58], [66]. Additionally, the repression degree of CaNACs in dehydrated roots versus dehydrated leaves was also lower by approximately 4-fold. For instance, CaNAC02 was the most highly down-regulated by dehydration in roots with a fold-change of only 6.2. In addition, CaNAC24 was deserved to be mentioned as this gene was induced (2.6-fold and 3.7-fold at 2 and 5 h after dehydration, respectively) in dehydrated roots but repressed (3-fold at 2 h of dehydration) in dehydrated leaves. It would be then interesting to study how CaNAC24 is involved in regulation of chickpea responses to drought. We hypothesize that under drought stress the up-regulation of CaNAC24 in roots might contribute to enhancement of root development, whereas its down-regulation in leaves might contribute to repression of leaf and/or shoot growth. These changes would enhance the adaptation of chickpea plants under limited water conditions.

Bottom Line:
Phylogenetic analysis of the chickpea and well-known stress-related Arabidopsis and rice NACs enabled us to predict several putative stress-related CaNACs.Nine-teen of the 23 CaNACs examined were found to be dehydration-responsive in chickpea roots and/or leaves in either ABA-dependent or -independent pathway.Our results have provided a solid foundation for selection of promising tissue-specific and/or dehydration-responsive CaNAC candidates for detailed in planta functional analyses, leading to development of transgenic chickpea varieties with improved productivity under drought.

ABSTRACTThe plant-specific NAC transcription factors (TFs) play important roles in regulation of diverse biological processes, including development, growth, cell division and responses to environmental stimuli. In this study, we identified the members of the NAC TF family of chickpea (Cicer arietinum) and assess their expression profiles during plant development and under dehydration and abscisic acid (ABA) treatments in a systematic manner. Seventy-one CaNAC genes were detected from the chickpea genome, including 8 membrane-bound members of which many might be involved in dehydration responses as judged from published literature. Phylogenetic analysis of the chickpea and well-known stress-related Arabidopsis and rice NACs enabled us to predict several putative stress-related CaNACs. By exploring available transcriptome data, we provided a comprehensive expression atlas of CaNACs in various tissues at different developmental stages. With the highest interest in dehydration responses, we examined the expression of the predicted stress-related and membrane-bound CaNACs in roots and leaves of chickpea seedlings, subjected to well-watered (control), dehydration and ABA treatments, using real-time quantitative PCR (RT-qPCR). Nine-teen of the 23 CaNACs examined were found to be dehydration-responsive in chickpea roots and/or leaves in either ABA-dependent or -independent pathway. Our results have provided a solid foundation for selection of promising tissue-specific and/or dehydration-responsive CaNAC candidates for detailed in planta functional analyses, leading to development of transgenic chickpea varieties with improved productivity under drought.